This invention relates to factors which inhibit neutrophil activity, including inhibition of neutrophil activation and adhesion of neutrophils to vascular endothelial cells.
Neutrophils are a class of white blood cells (leukocytes) that comprise an essential component of the host defense system against microbial invasion. In response to soluble inflammatory mediators released by cells at the site of injury, neutrophils migrate into tissue from the bloodstream by crossing the blood vessel wall. At the site of injury, activated neutrophils kill foreign cells by phagocytosis and by the release of cytotoxic compounds, such as oxidants, proteases and cytokines. Despite their importance in fighting infection, neutrophils themselves can promote tissue damage. During an abnormal inflammatory response, neutrophils can cause significant tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Alternatively, neutrophils that stick to the capillary wall or clump in venules may produce tissue damage by ischemia. Such abnormal inflammatory responses have been implicated in the pathogenesis of a variety of clinical disorders including adult respiratory distress syndrome (ARDS); ischemia-reperfusion injury following myocardial infarction, shock, stroke, and organ transplantation; acute and chronic allograft rejection; vasculitis; sepsis; rheumatoid arthritis; and inflammatory skin diseases (Harlan et al., 1990 Immunol. Rev. 114, 5).
Neutrophil adhesion at the site of inflammation involves at least two discrete cell-cell interactive events. Initially, vascular endothelium adjacent to inflamed tissue becomes sticky for neutrophils; neutrophils interact with the endothelium via low affinity adhesive mechanisms in a process known as xe2x80x9crollingxe2x80x9d. In the second adhesive step, rolling neutrophils bind more tightly to vascular endothelial cells and migrate from the blood vessel into the tissue. Neutrophil rolling along affected vascular segments and other initial low affinity contacts between neutrophils and the endothelium are mediated by a group of monomeric, integral membrane glycoproteins termed selectins. All three of the s-electins so far identified, that is L-selectin (LECAM-1, LAM-1) present on the surface of neutrophils, E-selectin (endothelial leukocyte adhesion molecule-1; ELAM-1) present on endothelial cells and P-selectin (granule membrane protein-140, GMP-140; platelet activation-dependent granule-external membrane protein, PADGEM; CD62) expressed on endothelial cells, have been implicated in neutrophil adhesion to the vascular endothelium (Jutila et al., 1989 J. Immunol 143, 3318; Watson et al., 1991 Nature 349, 164; Mulligan et al., J. Clin. Invest. 88, 1396; Gundel et al., 1991 J. Clin. Invest. 88, 1407; Geng et al., 1990 Nature 343, 757; Patel et al., 1991 J. Cell Biol. 112, 749). The counter-receptor for E-selectin is reported to be the sialylated Lewis X antigen (sialyl-Lewisx) that is present on cell-surface glycoproteins (Phillips et al., 1990 Science 250, 1130; Walz et al., 1990 Science 250, 1132; Tiemeyer et al., 1991 Proc. Natl. Acad. Sci. (USA) 88, 1138; Lowe et al., 1990 Cell 63, 475). Receptors for the other selectins are also thought to be carbohydrate in nature but remain to be elucidated.
The more stable secondary contacts between neutrophils and endothelial cells are mediated by a class of cell adhesion molecules known as integrins. Integrins comprise a broad range of evolutionarily conserved heterodimeric transmembrane glycoprotein complexes that are present on virtually all cell types. Members of the leukocyte-specific CD18 (xcex22) family of integrins, which include CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1; Mo-1; CR3) have been reported to mediate neutrophil adhesion to the endothelium (reviewed in Larson and Springer, 1990 Immunol Rev. 114, 181). Endothelial cell counter-receptors for these integrins are the intercellular cell adhesion molecules ICAM-1 and ICAM-2 for CD11a/CD18 and ICAM-1 for CD11b/CD18, respectively (Rothlein et al., 1986 J. Immunol. 137, 1270; Staunton et al., 1988 Cell 52, 925; Staunton et al., 1989 Nature 339, 61). The ICAMs are monomeric transmembrane proteins that are members of the immunoglobulin superfamily.
The activation of endothelial cells and neutrophils represents an important component of neutrophil-mediated inflammation. Factors that induce cell activation are termed agonists. Endothelial cell agonists, which include small regulatory proteins such as tumor necrosis factor (TNFxcex1) and interleukin-1 (IL-1xcex1), are released by cells at the site of injury. Activation of endothelial cells results in the increased surface expression of ICAM-1 (Staunton et al., 1988 Cell 52, 925) and ELAM-1 (Bevilacqua et al., 1987 Proc. Natl. Acad. Sci. (USA) 84, 9238). Raised levels of expression of these adhesive molecules on the surface of activated endothelial cells leads to the observed increased adhesivity of neutrophils for the vascular endothelium near sites of injury.
Activation of the neutrophil results in profound changes to its physiological state, including shape change, ability to phagocytose foreign bodies and release of cytotoxic substances from intracellular granules. Moreover, activation greatly increases the affinity of adhesive contacts between neutrophils and the vascular endothelium, perhaps through a conformational change in the CD11b/CD18 integrin complex on the neutrophil surface (Vedder and Harlan, 1988 J. Clin. Invest. 81, 676; Buyon et al., 1988 J. Immunol. 140, 3156). Factors that have been reported to induce neutrophil activation include IL-1xcex1, GM-CSF, G-CSF, MIP-1, IL-8 (IL-8=interleukin-8, GM-CSF=granulocyte/monocyte-colony stimulating factor, G-CSF=granulocyte-colony stimulating factor), and TNFxcex1, the complement fragment C5a, the microbe-derived peptide formyl-Met-Leu-Phe and the lipid-like molecules leukotriene B4 (LTB4) and platelet activating factor (Fuortes and Nathan, 1992, in Molecular Basis of Oxidative Damage by Leukocytes Eds Jesaitis, A. J. and Dratz, E. A. (CRC Press) pp. 81-90). In addition, phorbol esters (e.g., phorbol 12-myristate 13-acetate; PMA) represent a potent class of synthetic lipid-like neutrophil agonists. With the exception of PMA, these agonists have been reported to activate neutrophils by binding receptors on their surface. Receptors that are occupied by agonist molecules initiate within the neutrophil a cascade of events that ultimately results in the physiological changes that accompany neutrophil activation. This process is known as signal transduction. The lipid-like PMA likely effects neutrophil activation by passing through the plasma membrane at the cell surface and directly interacting with intracellular components (i.e., protein kinase) of the signal transduction machinery.
There exist two general classes of compounds that have been reported to down regulate the function of neutrophils, and these compounds have been reported to mitigate inflammation. One group of anti-inflammatory compounds is said to function as inhibitors of neutrophil activation, and presumably adhesion, by acting on components of the signal transduction machinery. A second class of anti-inflammatory compounds is said to block neutrophil infiltration into inflammatory foci by acting as direct inhibitors of the adhesive receptors that mediate contact between neutrophils and the vascular endothelium.
Many of the anti-inflammatory compounds currently used as therapeutics, including prostaglandins, catecholamines, and a group of agents known as non-steroidal anti-inflammatory drugs (NSAIDs), are believed to fall into the first category (Showell and Williams, 1989, in Immunopharmacology, eds. Gilman, S. C. and Rogers, T. J. [Telford Press, NJ] pp 23-63). For example, the enhanced adhesiveness observed for TNFa-activated neutrophils has been associated with decreased levels of a mediator of signal transduction, cyclic AMP (cAMP; Nathan and Sanchez, 1990 JCB 111, 2171). Exposure of neutrophils to prostaglandins and catecholamines has been correlated with elevated levels of intracellular cyclic AMP (cAMP; Showell and Williams, 1989). While the signal transduction inhibitors have been used extensively as anti-inflammatory therapeutic agents, they have several disadvantages including poor efficacy in acute inflammatory conditions, lack of specificity and undesirable side-effects such as gastric or intestinal ulceration, disturbances in platelet and central nervous system function and changes in renal function (Insel, 1990 in The Pharmacological Basis of Therapeutics, eds. Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P. [Pergamon, N.Y.], 8th Ed., pp. 638-681).
Glucocorticoids have long been recognized for their anti-inflammatory properties. Steroid-induced inhibition of neutrophils has been reported for several neutrophil functions, including adherence (Clark et al., 1979 Blood 53, 633-641; MacGregor, 1977 Ann. Intern. Med. 86, 35-39). The mechanisms by which glucocorticoids modulate neutrophil function are not well understood, but they are generally believed to involve the amplification or suppression of new proteins in treated neutrophils that play a key role in the inflammatory process (Knudsen et al., 1987 J. Immunol. 139, 4129). In particular, a group of proteins known as lipocortins, whose expression is induced in neutrophils by glucocorticoids, has been associated with anti-inflammatory properties (Flower, 1989 Br. J. Pharmacol. 94, 987-1015). Lipocortins may exert anti-neutrophil effects by interacting with sites on the neutrophil surface (Camussi et al., 1990 J. Exp. Med. 171, 913-927), but there is no evidence to suggest that the lipocortins act by directly blocking adhesive proteins on the neutrophil. Apart from their beneficial anti-inflammatory properties, glucocorticoids have been associated with significant side-effects. These include suppression of pituitary-adrenal function, fluid and electrolyte disturbances, hypertension, hyperglycemia, glycosuria, susceptibility to infection, ulcers, osteoporosis, myopathy, arrest of growth and behavioral disturbances (Insel, 1990).
A second class of anti-inflammatory compounds which are reported as direct inhibitors of neutrophil adhesion to the vascular endothelium have been described recently. Monoclonal antibodies that recognize and block ligand-binding functions of some of these adhesive molecules have proved to be effective in vivo inhibitors of neutrophil-mediated inflammation. In particular, monoclonal antibodies to the CD18 subunit of the CD18 integrin complexes (i.e., CD11a/CD18, CD11b/CD18 and CD11c/CD18) on the surface of neutrophils have been shown to prevent a variety of neutrophil-mediated tissue injury in animal models, including pulmonary edema induced by reperfusion (Horgan et al, 1990 Am. J. Physiol. 259, L315-L319), organ injury induced by hemorrhagic shock (Mileski et al, 1990 Surgery 108, 206-212), myocardial damage following ischemia/reperfusion (Winquist et al, 1990 Circulation III-701), edema and tissue damage following ischemia/reperfusion of the ear (Vedder et al, 1990 Proc. Natl. Acad. Sci. (USA) 87, 2643-2646), brain edema and death produced by bacterial meningitis (Tuomanen et al, 1989 J. Exp. Med. 170, 959-968), vascular injury and death in endotoxic shock (Thomas et al, 1991 FASEB J. 5, A509) and indomethacin-induced gastric injury (Wallace et al, 1991 Gastroenterology 100, 878-883).
Monoclonal antibodies directed to the CD11b subunit have been described. See, e.g:, Todd, R. F. et al., U.S. Pat. No. 4,840,793 (Jun. 20, 1989), Todd, R. F. et al., U.S. Pat. No. 4,935,234 (Jun. 19, 1990), Schlossman, S. F. et al., U.S. Pat. No. 5,019,648 (May 28, 1991) and Rusche, J. R. et al., International Application No. WO 92/11870 (Jul. 23, 1992). Monoclonal antibodies directed to CD18 subunit have been described. See, e.g., Arfors, K. E., U.S. Pat. No. 4,797,277 (Jan. 10, 1989), Wright, S. D. et al., European Patent Application No. 346,078 (Dec. 13, 1989), Law, M. et al., European Patent Application No. 438,312 (Jul. 24, 1991), Law, M. et al., European Patent Application No. 440,351 (Aug. 7, 1991), Wright, S. D. et al., U.S. Pat. No. 5,147,637 (Sep. 15, 1992) and Wegner, C. D. et al., European Patent Application No. 507,187 (Oct. 7, 1992).
Antibodies to other adhesive molecules have also been reported to have anti-inflammatory properties. Monoclonal antibodies that recognize the counter-receptor of CD11a/CD18 and CD11b/CD18, ICAM-1 have been reported to prolong cardiac allograft survival (Flavin et al, 1991 Transplant. Proc. 23, 533-534) and prevent chemically induced lung inflammation (Barton et al, 1989 J. Immunol. 143, 1278-1282). Furthermore, anti-selectin monoclonal antibodies have also been reported as efficacious in animal models of neutrophil-mediated inflammation. Monoclonal antibodies to L-selectin are reported to prevent neutrophil emigration into inflamed skin (Lewinshon et al., 1987 J. Immunol. 138, 4313) and inflamed ascites (Jutila et al., 1989 J. Immunol. 143, 3318; Watson et al., 1991 Nature 349, 164). Reports have also described inhibition of neutrophil influx into inflamed lung tissue by anti E-selectin monoclonal antibodies (Mulligan et al., 1991 J. Clin. Invest. 88, 1396; Gundel et al., 1991 J. Clin. Invest. 88, 1407). While the reports concerning activities of monoclonal antibodies to adhesive proteins are said to demonstrate the feasibility of using neutrophil adhesion inhibitors as anti-inflammatory agents, the utility of such monoclonal antibodies as therapeutics needs further evaluation.
Soluble adhesive receptors obtained by genetic engineering have been advanced as a further alternative approach as anti-inflammatory compounds. Soluble receptors, in which the transmembrane and intracellular domains have been deleted by recombinant DNA technology, have been reported to inhibit neutrophil adhesion to endothelial cells. The functional use of recombinant soluble adhesive molecules has been reported using CD11b/CD18 (Dana et al., 1991 Proc. Natl. Acad. Sci.(USA) 88, 3106-3110) and L-selectin (Watson et al., 1991, Nature 349:164-167).
Recently, a new class of anti-leukocyte compounds collectively termed leumedins has been reported. These compounds have been reported to block the recruitment in vivo of T lymphocytes and neutrophils into inflammatory lesions. The mechanism of action of the leumedins is unclear, but there is evidence that they do not function by blocking neutrophil activation (Burch et al., 1991 Proc. Natl. Acad. Sci. (USA) 88, 355). It remains to be determined if leumedins block neutrophil infiltration by direct interference with adhesive molecules.
It has been suggested that parasites survive in their host by modulating host immunity and inflammatory response though the mechanisms by which this occurs remains unclear (Leid, W. S., 1987, Veterinary Parasitology, 25: 147). In this regard, parasite-induced immunosuppression has been reported using certain rodent models (Soulsby et al., 1987, Immunol Lett. 16, 315-320).
Certain effects on neutrophils caused by materials isolated from parasites have been reported. For example, a protein isolated from the cestode, Taenia taeniaeformis, has been reported to inhibit chemotaxis and chemokinesis of equine neutrophils, as well as inhibit neutrophil aggregation (c. Suquet et al., 1984, Int""l J. Parasitol., 14: 165; Leid, R. W. et al., 1987, Parasite Immunology, 9: 195; and Leid, R. W. et al., 1987, Int""l J. Parasitol., 17: 1349). Peritoneal neutrophils from mice infected with the cestode, Echinococcus multiocularis, have been reported to lose their ability to migrate toward parasite antigens and nonspecific chemoattractants with increasing time of infection (Alkarmi, T. et al., Exptl. Parasitol., 1989, 69: 16). The nematode, Trichinella spiralis, has been reported to either excrete and/or secrete factors which inhibit chemotaxis and p-nitroblue tetrazolium reduction (i.e., release of oxidative metabolites) but enhance chemokinesis of human neutrophils (Bruschi, F. et al., 1989, Wiadomosci Parazytologiczne, 35: 391). The sera of humans infected with the nematode, Trichinella spiralis, has been reported to inhibit leukocyte chemotaxis and phagocytosis (Bruschi, F. et al., 1990, J. Parasitol., 76: 577). The saliva of the tick, Ixodes dammini, has been reported to inhibit neutrophil function (Ribeiro et al, 1990, Exp. Parasitol., 70, 382). A protein secreted by the cestode, Echinococcus granulosus, has been reported to inhibit human neutrophil chemotaxis (Shepard, J. C. et al., 1991, Mol. Biochem. Parasitol., 44: 81).
The present invention is directed to a neutrophil inhibitory factor (xe2x80x9cNeutrophil Inhibitory Factorxe2x80x9d or xe2x80x9cNIFxe2x80x9d) and to enriched compositions comprising Neutrophil Inhibitory Factor. Neutrophil Inhibitory Factor is a protein which is neither an antibody, a member of the integrin or selectin families nor a member of the immunoglobulin superfamily of adhesive proteins and which when isolated from a parasitic worm is a glycoprotein. Recombinant NIF""s produced by certain expression systems are not glycosylated. However, such non-glycosylated NIFs are considered to be within the scope of the present invention. A Neutrophil Inhibitory Factor of the present invention exhibits neutrophil inhibitory activity. Such neutrophil inhibitory activity may be demonstrated by its inhibition of at least one biological response in mammalian cells induced by activated neutrophils in an in vitro assay. Suitable assays for determining neutrophil inhibitory activity include those where inhibition of neutrophil activity is demonstrated by an assay which determines adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation or adhesion of neutrophils to plastic surfaces. In a preferred aspect the Neutrophil Inhibitory Factor comprises a protein present in and isolated from or substantially similar to a compound present in a parasitic worm, preferably canine hookworms, that inhibits neutrophil activity, particularly neutrophil adhesion to vascular endothelial cells. It is believed that certain isoforms of NIF are produced by the canine hookworm Ancylostoma caninum. This protein appears to act, at least in part, by inhibiting the process of neutrophil activation. A NIF has been demonstrated to be present in another parasitic worm, Toxocara canis. 
In view of the myriad conditions associated with undesired and/or abnormal inflammatory conditions which appear to be associated with neutrophil activity, there remains a need for potent, highly specific inhibitors of neutrophil function, in particular, adhesion to vascular endothelium, as a treatment for abnormal neutrophil-mediated inflammation. The present invention describes a potent and specific inhibitor of neutrophil activity, in particular the adhesion of neutrophils to vascular endothelial cells, derived from the hookworm (Ancylostoma caninum) and related species.
Among other factors, the present invention is based on our finding that the Neutrophil Inhibitory Factor of the present invention represents a pioneering step toward the development of a new generation of anti-inflammatory therapeutic products. This discovery will enable the first therapy for inflammatory disease based entirely on specific inhibition of the inflammatory response. The therapeutic advantages of this novel approach are realized through the specificity of Neutrophil Inhibitory Factor compared to current clinical treatment modalities such as steroids, catecholamines, prostaglandins, and nonsteroidal anti-inflammatory agents. The currently used class of therapeutic agents demonstrates poor efficacy and multiple adverse reactions due to generalized systemic effects that non-specifically target numerous biological processes in addition to the inflammatory process. Nonetheless, the existence of this extensive panel of anti-inflammatory agents, although suboptimal, and the total funds expended by the pharmaceutical industry in research in this area point to significant medical needs and suggests that the discovery of this novel and highly specific Neutrophil Inhibitory Factor will have important applications.
The inflammatory response results in clinical syndromes ranging from debilitating arthritis and asthma to life threatening shock. In view of the severity of these disorders, the vast number of afflicted individuals and the lack of suitable therapeutic intervention, the need for a breakthrough therapy represents a long felt need which has not been met. The Neutrophil Inhibitory Factor of the present invention represents such a breakthrough and provides the potential for a lifesaving therapy which is currently being sought throughout the international medical and pharmaceutical research communities.
The Neutrophil Inhibitory Factor can be isolated by preparing a soluble extract of the worm and fractionating it by chromatography on immobilized Concanavalin A, a molecular sieving matrix, and ceramic hydroxylapatite, and optionally, C4 reverse phase silica. Thus, according to another aspect, the present invention is directed to methods of isolating enriched compositions comprising Neutrophil Inhibitory Factor and the enriched compositions isolated by those methods. The factor can also be partially purified by preparative isoelectric focusing and chromatography on anion exchange media.
In one aspect, the present invention is directed to a composition enriched for Neutrophil Inhibitory Factor comprising a glycoprotein wherein the factor is isolated from a parasitic worm.
In another aspect, the present invention provides a composition enriched for Neutrophil Inhibitory Factor. In one preferred embodiment, the composition is isolated from a parasitic worm. Preferably the composition is enriched at least 200-fold for neutrophil inhibitory activity. Preferably the enriched composition is at least about 90% pure, more preferably, it is chromatographically pure.
According to one embodiment, the glycoprotein or Neutrophil Inhibitory Factor of the present invention is preferably acidic as determined by isoelectric focusing, having an isoelectric point of about 4.5, and preferably has a molecular weight in the range of about 38,000 to about 44,000 daltons as determined by laser-desorption time-of-flight mass-spectroscopy.
Preferably, the parasitic worm is a species selected from the phyla Platyhelminthes, Nematoda, Nematomorpha and Acanthocephala, more preferably Nematoda, and especially is isolated from a hookworm species such as those of the super family Ancylostomatidae.
The neutrophil inhibitory activity of the Neutrophil Inhibitory Factor of the present invention may be conveniently demonstrated by its inhibition of at least one biological response in mammalian cells induced by activated neutrophils in an in vitro assay. Suitable assays include those which determine adhesion of neutrophils to vascular endothelial cells or to plastic surfaces, release of hydrogen peroxide by neutrophils or homotypic neutrophil aggregation. Suitable Neutrophil Inhibitory Factors exhibit an IC50 of about 500 nM or less, more preferably less than 100 nM.
According to a further aspect of the present invention, methods of preparing biologically active Neutrophil Inhibitory Factor are provided. These methods comprise culturing host cells containing an expression vector which encodes a gene for a glycoprotein having neutrophil inhibitory activity isolated from a hookworm, preferably a canine hookworm, which has apparent molecular weight of about 38,000 to about 44,000 daltons as determined by laser-desorption time-of-flight mass spectrometry, and to the recombinant Neutrophil Inhibitory Factor produced according to those methods.
Also encompassed within the scope of the invention are isolated nucleic acid molecules, preferably DNA, which code for Neutrophil Inhibitory Factor, vectors, (including cloning and expression vectors) which contain the nucleic acid molecule and host cells transformed with such vectors.
The present invention also provides methods of preparing recombinant Neutrophil Inhibitory Factor using a nucleic acid molecule encoding the Neutrophil Inhibitory Factor. The nucleic acid molecule is expressed in a cultured host cell transformed with a vector containing the nucleic acid molecule operably linked to control sequences recognized by the host cell.
In a further aspect, the present invention is directed to antibodies against Neutrophil Inhibitory Factor, including monoclonal antibodies and hybridomas which produce the monoclonal antibodies, and to immunoassays using the antibodies.
The invention is also directed to pharmaceutical compositions comprising a therapeutically effective amount of Neutrophil Inhibitory Factor and a pharmaceutically acceptable carrier, and the methods of using these pharmaceutical compositions to treat inflammatory conditions, especially to prevent or decrease inflammatory responses. In particular, such pharmaceutical compositions may comprise Neutrophil Inhibitory Factor and a pharmaceutically acceptable carrier, wherein the Neutrophil Inhibitory Factor interacts with neutrophils to inhibit their activity and prevents and/or decreases inflammatory responses in a mammalian host caused by neutrophils when a therapeutically effective amount of Neutrophil Inhibitory Factor is administered.
According to a further aspect, the present invention is directed to methods of isolating NIF-like proteins and to NIF-like proteins so isolated. These NIF-like proteins may be isolated by preparing a genomic or cDNA library from a source, whether animal, bacterial, fungal or viral, which is suspected of containing Neutrophil Inhibitory Factor, hybridizing oligonucleotide probes sufficiently complementary to hybridize to a nucleic acid encoding a NIF to the library and isolating nucleic acid sequences which hybridize to the probes. The nucleic acid sequence can then be cloned and expressed. Alternatively NIF-like proteins may be isolated which include a protein which is encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to a probe having at least about 12 nucleotides which is complementary to a portion of nucleic acid sequence encoding a NIF, in one preferred aspect the sequence of FIG. 8.
Other features and advantages of the invention will be apparent from the following descriptions of the preferred embodiments and from the claims.